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Ischemic Preconditioning: Triggering Role of Nitric Oxide-Derived Oxidants in Isolated Hearts

Novalija, Enis; Hogg, Neil; Kevin, Leo G.; Camara, Amadou K.S.; Stowe, David F.

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Journal of Cardiovascular Pharmacology: November 2003 - Volume 42 - Issue 5 - p 593-600
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It is well established that cardiac injury upon aerobic reperfusion after prolonged global (index) ischemia has an oxidative component. There is a significant body of evidence, based on the effects of putative antioxidants (particularly the non-physiological thiol, 2-mercaptopropionyl glycine (2-MPG)), that ischemic preconditioning may also involve the partial reduction of molecular oxygen, and that oxidants generated from this process may trigger or mediate cardiac protection in both the early-phase and late-phase preconditioning. 1,2 Mechanistic studies using hypoxia/reoxygenation of isolated myocytes have implicated a signaling role of oxidants in acetylcholine-mediated preconditioning. 3 Vanden Hoek et al, 4 using the same model, observed the oxidation of dichlorofluorescein and dihydroethidium during hypoxic preconditioning, and concluded that increases in mitochondrial superoxide formation during hypoxia initiate the preconditioning process.

The interaction between partially reduced forms of molecular oxygen and the free radical, nitric oxide (NO), appears to be a key event in controlling and redirecting the chemical behavior of oxygen-derived oxidants. 5 For example, the diffusion-limited reaction between nitric oxide and superoxide leads to the formation of peroxynitrite, an oxidant with differing biologic chemistry than its precursor radicals. 6 Although there is strong evidence for a role of nitric oxide in late-phase preconditioning, 2 the effect of nitric oxide in classic preconditioning is more controversial. Nakano et al, 7 using an isolated rabbit heart model, reported that S-nitroso-N-acetylpenicillamine (SNAP), an S-nitrosothiol often employed as a putative NO donor, mimicked ischemic preconditioning. In this study, 2-MPG blocked SNAP-induced cardioprotection and increased infarct size, suggesting that exogenous NO induces preconditioning via MPG-sensitive oxidants. NG-nitro l-arginine methyl ester (l-NAME) failed to block ischemic preconditioning however; thus a contribution of endogenous NO to classic preconditioning was not demonstrated in this model. In contrast, Vegh et al 8 demonstrated antagonistic effects of l-NAME to preconditioning in an open-chest dog model. Vanden Hoek et al demonstrated that the presence of a nitric oxide synthase inhibitor increased dichlorofluorescein fluorescence during hypoxic preconditioning in an isolated myocyte model, 4 and more recently demonstrated that NOS inhibition reduced the effectiveness of preconditioning hypoxia in this same model. 9

It was recently suggested, based on formation of nitrotyrosine in the coronary effluent of a Langendorff-perfused rat heart preparation, that peroxynitrite can be formed both during the reperfusion period and during the ischemic preconditioning periods. 10 In addition it was demonstrated that ischemic preconditioning reduced the level of nitrotyrosine detected during subsequent ischemia/reperfusion. While the study by Czonka et al 10 elegantly demonstrates that nitration chemistry occurs upon reperfusion of ischemic myocardium, and that the extent of nitration is related to the severity of the injury, it does not establish a cause-and-effect relationship between the formation of nitrating agents and preconditioning.

In this study we examined the relationship between oxidants generated during ischemic preconditioning and the extent of protection. We have modulated oxidant levels by the use of antioxidants and NGnitro l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthase. As the primary indication of oxidant production we have chosen to monitor the formation of diotyrosine from the oxidation of tyrosine. We have recently demonstrated the formation of diotyrosine during ischemia/reperfusion, and its ablation in response to anesthetic preconditioning. 11

We show here that during ischemic preconditioning and during reperfusion after index ischemia, in a Langendorf-perfused guinea pig heart, an oxidant is formed that is strong enough to initiate tyrosine dimerization. In addition we demonstrate that the presence of either an antioxidant cocktail (consisting of superoxide dismutase, catalase, and glutathione(SCG)) or l-NAME during ischemic preconditioning can abolish diotyrosine formation during the preconditioning period, enhance diotyrosine formation on reperfusion after index ischemia, and significantly reduce the cardioprotective effects of ischemic preconditioning. These data support the hypothesis that the formation of nitric oxide-derived oxidants during ischemic preconditioning is causally related to myocardial adaptation to reperfusion injury.


Langendorff Heart Preparation

The investigation conformed to the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health No. 85-23, revised 1996). Prior approval was obtained from the Medical College of Wisconsin animal studies committee. The preparation has been described in detail previously. 12 Guinea pigs (n = 68) were prepared by the Langendorff method and perfused via the aortic root at 55 mm Hg with a 37°C oxygenated, modified Krebs-Ringer's solution as described previously. 12

Left ventricular pressure (LVP), spontaneous heart rate (HR), atrial-ventricular (AV) conduction time, and coronary flow (CF) were measured continuously. Coronary sinus effluent was collected by placing a small catheter into the right ventricle through the pulmonary artery after ligating both venae cavae. Coronary sinus venous PO2 tension was also measured continuously in-line with an oxygen Clark type electrode (Model 203B, Instech, Plymouth Meeting, PA). Percent oxygen extraction (% O2E) was calculated as 100 · [(PO2a−PO2v)/PO2a)]; myocardial oxygen consumption (MVO2) as CF/g · (PO2a-PO2v) · 24 μL O2/mL at 760 mm Hg; and cardiac efficiency as systolic-diastolic LVP · HR/MVO2.

Effluent was spot collected during reperfusion and frozen for later analysis of creatine kinase (Creatine Kinase Flex™ reagent cartridge, Dade Behring Dimension, Newark, DE; sensitivity >10 U/L). If ventricular fibrillation (VF) occurred, a 0.25-mL bolus of lidocaine (250 μg) was administered immediately via the aortic cannula. Data were collected only from hearts naturally in, or converted to, sinus rhythm. Infarct size was determined by the 2,3,5-triphenyltetrazolium chloride (TTC) staining method and expressed as a percentage of the total heart weight. 12

Detection of Dityrosine using Spectrofluorometry

Formation of diotyrosine was analyzed by measuring fluorescence emission, λex 320 nm and λem 410 nm, at room temperature using a spectrophotofluorometer (Perkin Elmer model LS 50B, Beaconsfield, Buckinghamshire, UK). Dityrosine concentration measured by HPLC is linearly related to fluorescence intensity (r2 > 0.99); the detection limit for dityrosine is reported as 0.05 μM. 13 Reaction of l-tyrosine with authentic peroxynitrite to produce dityrosine in Krebs-Ringer's buffer occurs within 1 minute and the product is stable in room air for over 1 hour. 13 Collected effluent samples were kept at 3°C for no more than 1 hour before analysis. The method was tested using dityrosine generated from the reaction between peroxynitrite and l-tyrosine. Both authentic and decomposed peroxynitrite were prepared by the method described by Villa et al. 14


Figure 1 shows the experimental design. There were 6 groups (n = 10 hearts each group) subjected to ischemia and one control group not subjected to ischemia (n = 8, data not shown). This group will be referred to as the non-ischemic control. Each experiment lasted 200 minutes beginning 30 minutes after equilibration. Hearts were assigned randomly into 1 of the 7 groups. In 3 ischemic preconditioning (IPC) groups, hearts were exposed to two, 2-minute periods of ischemia (ie, preconditioning stimuli) separated by 6 minutes of reperfusion, ending 20 minutes before 30 minutes of index ischemia and 120 minutes of reperfusion. Antioxidants, or the NO synthesis inhibitor l-NAME, were given from 5 minutes before the preconditioning stimuli until 5 minutes after the second preconditioning stimulus with a 15-minute washout period before the onset of index ischemia. Added to the perfusate were 50 U/mL superoxide dismutase, 50 U/mL catalase, and 0.5 mM glutathione (ie, SCG) or alternatively, l-NAME, 100 μM (groups IPC+SCG and IPC+L-NAME). Two additional groups of hearts were pretreated with the combination of the antioxidants alone (SCG) or with l-NAME alone, to rule out any direct effects of these drugs.

Experimental protocols for 6 randomized groups of guinea pig hearts. Ischemic preconditioning (IPC) stimuli were elicited by brief ischemia for two, 2-minute periods of ischemia (ie, preconditioning stimuli) separated by 6 minutes of reperfusion. Combination of antioxidants, superoxide dismutase (50 U/mL), catalase (50 U/mL), and glutathione (0.5 mM) (SCG), or the NO• synthesis inhibitor NGnitro l-arginine methyl ester (L-NAME, 100 μM), were given from 5 minutes before the preconditioning stimuli until 5 minutes after the second preconditioning stimulus with a 15-minute washout period before the onset of index ischemia.

Coronary flow responses to bolus adenosine (0.2 mL of 200 mM stock), 100 μM nitroprusside, and 10 nM bradykinin were tested at the end of reperfusion. For dityrosine measurements, coronary effluent samples (2.7 mL) were collected during baseline (at 15 minutes), over 1 minute after each preconditioning stimuli (at 23 and 31 minutes), 1 minute before index ischemia (at 50 minutes), each minute during the first 5 minutes of reperfusion (at 81–85 minutes), and at the tenth minute of reperfusion (at 90 minutes). For creatine kinase measurements, 2 mL effluent was collected 1 minute before index ischemia and at 1, 5, and 15 minutes of reperfusion.

Statistical Analysis

All data were expressed as mean ± standard error of the means (SEM). Within-group data (time effect) for a given variable were compared with a baseline control period (at 15 minutes) by Duncan's comparison of means test whenever univariate analysis of variance for repeated measures was significant (Super ANOVA 1.11® software for Macintosh® from Abacus Concepts, Inc, Berkeley, CA). Among-group data (treatment effect) at specific time points (at 15, 23, 30, 81, 110, 140, 200 minutes) were analyzed using multivariate analysis for repeated measures. If F values were significant (P < 0.05), post-hoc comparisons of means compared with the baseline (at 15 minutes) control period (Student's t test with Duncan's adjustment for multiplicity) were used to differentiate treatment groups. Infarct size and creatine kinase were similarly analyzed statistically. The incidence of VF versus sinus rhythm was determined by χ2 analysis and differences in VF duration were determined by unpaired t tests. Differences among means were considered statistically significant when P < 0.05.


Baseline Measurements

There were no differences in baseline values (at 15 minutes) for all cardiac variables and effluent measurements among the 6 groups subjected to ischemia. For the non-ischemic group, there were no significant changes over time (15–200 minutes) for any variable (data not shown).

Mechanical Effects

Systolic-diastolic (developed) LVP was not different among groups before index ischemia (Fig. 2) but was reduced in all groups after ischemia compared with the non-ischemic control (data not shown). Developed LVP was significantly greater in the IPC group than in the control group. The presence of the antioxidant cocktail (SCG group) or l-NAME, during the ischemic preconditioning period abolished the protective effect of preconditioning. When these agents were given alone, no direct effects were observed. End diastolic LVP (Table 1) was lower in the IPC group than in the other groups at 81 minutes (onset of reperfusion) and 200 minutes (end of reperfusion) indicating that the beneficial effect of IPC on developed LVP was due to attenuated diastolic contracture.

End Diastolic (ED) LVP (mm Hg), Percent Oxygen Extraction (% O2), and Efficiency Index (EI) (mmHg · beat · g−1 · 100 nL O2−1) after Index Ischemia in CONTROL, IPC, SCG, IPC + SCG, L-NAME, and IPC + L-NAME groups
Time course for developed (systolic-diastolic) LVP before, during, and after index ischemia in CONTROL and ischemic preconditioned groups (IPC), with and without a combination of antioxidants (SCG) or l-NAME. Developed LVP was least reduced in the IPC-treated groups on reperfusion with no differences among the remaining groups. Values are means ± SEM; n = 10 for each group. Statistical symbol for P < 0.05 is * versus CONTROL.

Metabolic Effects

Figure 3 shows that myocardial oxygen consumption (MVO2) fell below baseline in each group after index ischemia, but was significantly higher in the IPC group compared with control. Again, the presence of SCG or l-NAME during the preconditioning period abolished this protective effect of IPC, but when these agents were given alone, no direct effects were observed. Percent oxygen extraction at 120 minutes reperfusion was significantly lower for the IPC group than for all other groups (Table 1).

Time course for myocardial oxygen consumption (MVO2, μL O2 · g−1 · min−1) before, during, and after index ischemia in CONTROL and IPC hearts, with and without a combination of antioxidants (SCG) or l-NAME. MVO2 was greater during reperfusion after IPC compared with other groups. Values are means ± SEM; n = 10 for each group. Statistical symbol for P < 0.05 is * versus CONTROL.

The cardiac efficiency index (mm Hg · beat)/(100 nL O2 · g−1) calculated from the above data, shown in Table 1, mirrors the above observations.

Table 2 shows that coronary flow in mL · g−1 · min−1 was higher throughout reperfusion in the IPC group than in any other group. The post-ischemic reactive-flow increase during the initial 2-minute reperfusion period was apparent only in the IPC group. Flow was lowest at the first minute of reperfusion in the control group and was similarly lower in all but the IPC group after 120 minutes of reperfusion. Coronary flow responses to adenosine, nitroprusside, and bradykinin, respectively, after 120 minutes reperfusion were significantly higher after IPC and were reduced by the presence of SCG or l-NAME (Table 2).

Coronary Flow (ml · min−1 · g−1) before and after Index Ischemia, and Responses to Adenosine (ADE), nitroprusside (NP), and Bradykinin (BK), Respectively, in CONTROL, IPC, SCG, IPC + SCG, L-NAME, and IPC + L-NAME groups

Electrical Effects

For all groups before index ischemia (at 50 minutes), and after reperfusion (at 200 minutes), there were no differences in heart rate (253 ± 3 and 255 ± 3 beats/min, respectively) or AV conduction time, (74 ± 3 and 75 ± 2 milliseconds, respectively); these values were averaged for all groups. The only dysrhythmia observed on reperfusion was ventricular fibrillation (VF), which occurred in all ischemic groups. The incidence of VF for each group, including repeat VF in a given heart, are shown in Table 3. When VF occurred, its onset was within 1 minute of reperfusion except in the IPC group, where the onset was much later, at 5.3 ± 0.3 minutes (P < 0.05).

Incidence of Ventricular Fibrillation (VF) and Concentration of Creatine Kinase (units · L−1) during Reperfusion in CONTROL, IPC, SCG, IPC + SCG, L-NAME, and IPC + L-NAME groups

Infarct Size

The inhibition of the functional and metabolic cardioprotective effects of IPC by antioxidants and l-NAME were accompanied by a significant increase in infarct size (Fig. 4). Myocardial infarct size was significantly smaller in the IPC group and increased to control levels in the SCG and l-NAME groups. Creatine kinase release into coronary effluent (Table 3) paralleled infarct size and was not detectable before index ischemia (at 15 and 50 minutes) in any group.

Myocardial infarct size expressed as a percentage of total heart weight in hearts receiving a combination of antioxidants (SCG) or l-NAME in the presence or absence of ischemic preconditioning. Note the large protection against infarction by IPC compared with each other group. Values are means ± SEM; n = 10 for each group. Statistical symbol for P < 0.05 is * versus CONTROL.

Oxidant Formation

Diotyrosine fluorescence (Fig. 5) was detected after the 2 ischemic preconditioning stimuli in the IPC group. The level of dityrosine was significantly greater after the second stimulus (P < 0.05). Dityrosine fluorescence was not detected during the preconditioning stimuli when bracketed either by antioxidants or l-NAME. After index ischemia, dityrosine fluorescence was observed in the control group, and it was markedly decreased in the IPC group. The presence of either antioxidants or l-NAME during the preconditioning stimuli restored dityrosine fluorescence on reperfusion to control levels. The changes in dityrosine fluorescence could not be explained by changes in coronary flow. Relative fluorescence normalized to coronary flow was significantly lower after IPC compared with control at 1 minute of reperfusion.

Release of fluorescent diotyrosine into the coronary effluent immediately after each preconditioning ischemia stimulus and on initial reperfusion after index ischemia in CONTROL and IPC hearts, with and without a combination of antioxidants (SCG) or l-NAME. Values are expressed as the change in relative units of fluorescence from the baseline value measured in effluent before treatments. Note the change in the time scale. Diotyrosine concentration increased only in the IPC group after preconditioning stimuli and increased least in the IPC group on reperfusion after index ischemia. Values are means ± SEM; n = 10 for each group. Statistical symbol for P < 0.05 is * versus CONTROL.


In this study we used a model of the intact beating heart to examine the relationship between the cardioprotective effects of ischemic preconditioning, manifested as improved cardiac rhythm, perfusion, mechanical, and metabolic function, and oxidant formation during ischemic preconditioning and on reperfusion after index ischemia.

In agreement with previous reports, brief periods of ischemia 15 to 20 minutes before index ischemia resulted in significant cardioprotection. 15,16 Protection was apparent in functional, metabolic, and histologic parameters. The presence of an antioxidant cocktail consisting of SCG during the preconditioning period inhibited the cardioprotective effect. This is in general agreement with other studies showing an inhibitory effect of thiolic antioxidants and oxidant scavenging enzymes. 1,17–19 The nitric oxide synthase inhibitor l-NAME also inhibited cardioprotection when present during the preconditioning period. This is in contradiction to Nakano et al 7 who reported no effect of nitric oxide synthase inhibitors in a rabbit model of preconditioning, but is in general agreement with the study by Vegh et al 8 and Lochner et al. 20 To measure oxidant formation during both the ischemic preconditioning period and the reperfusion period after index ischemia, the hearts were perfused with l-tyrosine, and dityrosine was monitored in the coronary effluent. Dityrosine formation was observed after the 2 ischemic preconditioning stimuli and during reperfusion. Ischemic preconditioning reduced the level of dityrosine observed upon reperfusion after index ischemia, and there was a correspondence between the amount of dityrosine observed upon reperfusion and the extent of injury. Importantly, the formation of dityrosine during the preconditioning period was abolished in the presence of either antioxidants or l-NAME, but conversely this treatment led to an enhancement of the production of dityrosine upon reperfusion after index ischemia. These observations indicate that an oxidant is formed during ischemic preconditioning that is strong enough to oxidize tyrosine to a tyrosyl radical, consequently generating dityrosine. The oxidant can be scavenged, or its formation inhibited, by antioxidants (SCG), and its presence is dependent upon the activity of nitric oxide synthase. The most likely candidate for this oxidant is peroxynitrite, the product of the reaction between nitric oxide and superoxide that has been shown to oxidize tyrosine to dityrosine in vivo.

Kanzik's group have shown that the isolated rat heart can be preconditioned with chemically synthesized peroxynitrite;21,22 however, these studies were conducted at pH 8.4, where peroxynitrite has a half time of about 20 to 30 seconds and no control of pre-decomposed peroxynitrite was performed. It is not clear therefore, whether these effects were due to peroxynitrite per se or a decomposition product of peroxynitrite. Precedence for peroxynitrite formation during preconditioning was provided by Csonka et al, 10 who measured nitrotyrosine in effluent of isolated rat hearts. They demonstrated the formation of nitrotyrosine during the ischemic preconditioning period as well as a preconditioning-dependent reduction of tyrosine nitration on reperfusion after index ischemia. However, these investigators did not assess if the nitrating agent formed during IPC was an important mediator of cardioprotection. In this study, we examined if a causal relationship exists between oxidant formation during the ischemic preconditioning period and cardioprotection afforded upon reperfusion. We observed that reduction of oxidant formation during the ischemic preconditioning period, by administration of antioxidants or l-NAME, enhanced oxidant formation upon reperfusion. While it is difficult to conclusively demonstrate the formation of peroxynitrite, the data presented here, in combination with that shown by Csonka et al 10 strongly indicate that peroxynitrite is formed during the ischemic preconditioning period.

One difference between the report of Csonka et al 10 and the data presented here, is that in the former study the level of nitrotyrosine decreased after each ischemic preconditioning stimulus, whereas in this study dityrosine was higher after the second pulse than the first. It is not clear, however, that this represents a mechanistic difference in the 2 systems. It has been demonstrated that the ratio of dityrosine to nitrotyrosine formation from both peroxynitrite and from the simultaneous formation of nitric oxide and superoxide is highly variable and depends, among other things, on the rate of peroxynitrite generation and the concentration of carbon dioxide in the perfusate. 23,24

The ability of l-NAME to reduce the efficacy of preconditioning indicates that nitric oxide is an important intermediate in the pathway to myocardial adaptation. It is not clear why other studies have not observed an effect of NOS inhibition in similar models, but this may be related to the concentration of inhibitor used. For example, Weselcouch et al 25 used a concentration of l-NAME (30 μM) that was sufficient to inhibit vasorelaxation in response to acetylcholine. However, this functional end point may not indicate complete inhibition of nitric oxide synthesis, as the rapid scavenging of nitric oxide by superoxide will create a threshold level of nitric oxide synthase that must be exceeded before relaxation will occur. As intracellular arginine concentration is usually over 100 μM, it is possible that low levels of l-NAME are not completely inhibiting the enzyme.

Previous studies of hypoxia-reoxygenation injury in isolated myocytes have indicated a role for oxidant production in the protection afforded by hypoxic preconditioning. Vanden Hoek et al 4 observed rapid and robust oxidation of dichlorofluorescein hydrochloride (DCFH) to its fluorescent oxidation product upon initiating hypoxia in isolated myocytes. Reintroduction of oxygen after 10 minutes caused a reduction in fluorescence, and these data were interpreted to mean that oxidant formation occurred during hypoxia and was curtailed upon reperfusion. Ten minutes of preconditioning hypoxia were sufficient in this model to significantly protect the cells from reoxygenation injury after 1 hour of hypoxia, and evidence using thiol and dithiocarbamate interventions suggested hydrogen peroxide, derived from mitochondrial superoxide, was a major contributor to myocyte protection. Interestingly, they demonstrated that an inhibitor of nitric oxide synthase increased fluorophore formation. A recent study by this same group, using the same model, indicated that l-NAME significantly attenuates preconditioning while increasing apparent oxidant formation (as measured by DCF fluorescence). 9 It can be concluded from these studies that ischemic preconditioning is mediated by both superoxide and nitric oxide and not by superoxide alone. The enhancement of DCF fluorescence by l-NAME observed by Vanden Hoek et al 4 strongly suggests that significant interaction between nitric oxide and superoxide is occurring, as decreased nitric oxide formation should enhance superoxide dismutation leading to increased hydrogen peroxide formation and hence increased DCF fluorescence. It is highly likely therefore, that under these conditions peroxynitrite is formed.

The data presented here indicate that an oxidant with the characteristics of peroxynitrite is formed during ischemic preconditioning, and that the prevention of formation of this oxidant by l-NAME abolishes the preconditioning effect. It cannot be firmly concluded that peroxynitrite mediates preconditioning, as the SCG treatment could act at many levels (prevention of oxidant formation, oxidant scavenging, or repair of oxidative damage). However, a role of peroxynitrite in ischemic preconditioning is a reasonable inference. Definitive proof awaits the development of a highly specific peroxynitrite scavenger.

The mechanisms responsible for protection in cardiac preconditioning are not completely understood. Our data are consistent with the model proposed by Lebuffe et al 9 using isolated myocytes. This pathway involves the formation of mitochondrial superoxide, and likely involves subsequent activation of protein kinase C and the mitochondrial KATP channel. Future work is required to identify the oxidant sensing machinery that is ultimately responsible for initiating the cellular and subcellular changes that characterize the preconditioned heart.


The authors thank Cathleen Berglund, Mary Lorence-Hanke, James Heisner, and Anita Tredeau for their valuable contributions to this study (all of Medical College of Wisconsin, Milwaukee, Wisconsin).


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contractile function; dityrosine; free radicals; ischemic preconditioning; isolated guinea pig heart; nitric oxide

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